The human lung mucosa drives differential Mycobacterium tuberculosis infection outcome in the alveolar epithelium.

Mycobacterium tuberculosis (M.tb) is deposited into the alveolus where it first encounters the alveolar lining fluid (ALF) prior contacts host cells. We demonstrated that M.tb-exposure to human ALF alters its cell surface, driving better M.tb infection control by professional phagocytes. Contrary to these findings, our results with non-professional phagocytes alveolar epithelial cells (ATs) define two distinct subsets of human ALFs; where M.tb exposure to Low (L)-ALF or High(H)-ALF results in low or high intracellular bacterial growth rates in ATs, respectively. H-ALF exposed-M.tb growth within ATs was independent of M.tb-uptake, M.tb-trafficking, and M.tb-infection induced cytotoxicity; however, it was associated with enhanced bacterial replication within LAMP-1+/ABCA1+ compartments. H-ALF exposed-M.tb infection of ATs decreased AT immune mediator production, decreased AT surface adhesion expression, and downregulated macrophage inflammatory responses. Composition analysis of H-ALF vs. L-ALF showed H-ALF with higher protein tyrosine nitration and less functional ALF-innate proteins important in M.tb pathogenesis. Replenishment of H-ALF with functional ALF-innate proteins reversed the H-ALF-M.tb growth rate to the levels observed for L-ALF-M.tb. These results indicate that dysfunctionality of innate proteins in the H-ALF phenotype promotes M.tb replication within ATs, while limiting inflammation and phagocyte activation, thus potentiating ATs as a reservoir for M.tb replication and survival.

Exposure to human alveolar lining fluid enhances Mycobacterium bovis BCG vaccine efficacy against Mycobacterium tuberculosis infection in a CD8+ T-cell-dependent manner.

Current tuberculosis (TB) treatments include chemotherapy and preventative vaccination with Mycobacterium bovis Bacillus Calmette-Guérin (BCG). In humans, however, BCG vaccination fails to fully protect against pulmonary TB. Few studies have considered the impact of the human lung mucosa (alveolar lining fluid (ALF)), which modifies the Mycobacterium tuberculosis (M.tb) cell wall, revealing alternate antigenic epitopes on the bacterium surface that alter its pathogenicity. We hypothesized that ALF-induced modification of BCG would induce better protection against aerosol infection with M.tb. Here we vaccinated mice with ALF-exposed BCG, mimicking the mycobacterial cell surface properties that would be present in the lung during M.tb infection. ALF-exposed BCG-vaccinated mice were more effective at reducing M.tb bacterial burden in the lung and spleen, and had reduced lung inflammation at late stages of M.tb infection. Improved BCG efficacy was associated with increased numbers of memory CD8+ T cells, and CD8+ T cells with the potential to produce interferon-γ in the lung in response to M.tb challenge. Depletion studies confirmed an essential role for CD8+ T cells in controlling M.tb bacterial burden. We conclude that ALF modifications to the M.tb cell wall in vivo are relevant in the context of vaccine design.

Pulmonary defense and the human cathelicidin hCAP-18/LL-37.

Antimicrobial peptides form an important component of the innate immune system. The cathelicidin family, a key member of the antimicrobial peptide defenses, has been highly conserved throughout evolution. Though widespread in mammals, there is currently only one identified human example, hCAP-18/LL-37. The cathelicidins have been found to have multiple functions, in addition to their known antimicrobial and lipopolysaccharide-neutralizing effects. As a result, they profoundly affect both innate and adaptive immunity. Currently, antimicrobial peptides are being evaluated as therapeutic drugs in disease states as diverse as oral mucositis, cystic fibrosis, and septic shock. One such peptide, the cathelicidin hCAP-18/LL-37, is reviewed in detail in the context of its role in lung physiology and defense.

Sequential processing of human ProIL-1beta by caspase-1 and subsequent folding determined by a combined in vitro and in silico approach.

PURPOSE: Interleukin-1beta is a multifunctional cytokine produced by activated monocytes and macrophages that requires caspase-1 (IL-1 converting enzyme/ICE) to process the 31kDa inactive precursor protein to the biologically active 17kDa peptide. This activation event involves ICE cleavage at Asp27 (site 1) and Asp116 (site 2). To address the sequential processing at ICE cut sites we combined in vitro analysis and molecular modeling to investigate the sequence of molecular events. METHODS: Pulse chase labeling followed by immunoprecipitation of IL-1beta in activated human monocyte lysates demonstrated sequential cutting by ICE at site 1 before site 2 in vitro. To corroborate these findings, we constructed a homology model of proIL-1beta after the crystal structure of another ICE substrate, human alpha1-antitrypsin (23% sequence identity). RESULTS: Comparative modeling revealed that site 1 on proIL-1beta is accessible to ICE but site 2 is not. Molecular dynamics simulations following ICE cleavage at site 1 and removal of the 3kDa amino-terminal fragment, rendered site 2 accessible to ICE. CONCLUSIONS: The close agreement between the in vitro and modeled behavior of IL-1beta support our contention that IL-1beta may be structurally related to alpha1-antitrypsin and also predicts that proIL-1beta requires sequential processing for activation. These findings may facilitate the development of novel pharmacological agents that control posttranslational proIL-1beta modification, thereby preventing excessive production of this potent inflammatory cytokine.

Interleukin-1beta costimulates interferon-gamma production by human natural killer cells.

Natural killer (NK) cells are an early source of immunoregulatory cytokines during the innate immune response to viruses, bacteria, and parasites. NK cells provide requisite IFN-gamma to monocytes for the elimination of obligate intracellular pathogens. IL-1beta is a pro-inflammatory cytokine produced by monocytes (i.e. a monokine) during the early immune response to infection, but its role in promoting human NK cell IFN-gamma production is unknown. The current study examines the ability of the monokine IL-1beta, plus IL-12, to costimulate IFN-gamma production by resting CD56(bright) and CD56(dim) human NK cell subsets. CD56(bright) NK cells stimulated with IL-1beta plus IL-12 produced abundant IFN-gamma protein, while little IFN-gamma was produced in identical cultures of CD56(dim) cells. In addition, upon activation with IL-1beta, CD56(bright) NK cells exhibited considerably greater phosphorylation of extracellular signal-regulated kinases p42/44 as compared to CD56(dim) NK cells. Quantitative PCR analysis showed brisk induction of IFN-gamma gene expression following costimulation with IL-1beta plus IL-12 in CD56(bright) NK cells, but intracellular flow cytometry revealed that only a fraction (42+/-2.3%) of CD56(bright) NK cells account for this high IFN-gamma production. These data suggest that the monokine IL-1beta is a potent costimulus of IFN-gamma production by a subset of NK cells following infectious insult.

BAL and serum IgG levels in healthy asymptomatic HIV-infected patients.

OBJECTIVES: To determine if the increased susceptibility to bacterial infection in asymptomatic HIV-infected patients is associated with decreased total IgG or IgG2 levels in lung epithelial lining fluid. BACKGROUND: A decrease in lung IgG levels or subtypes has been proposed as contributing to the increased risk of bacterial lung infections in HIV-infected patients. Previous studies measuring lung lavage IgG concentrations have been inconsistent. METHODS: Twenty-three HIV patients and 25 control subjects underwent BAL. Both patient groups were of similar age, and had similar pulmonary function studies and body mass index. Smokers were equally represented in both groups, and the majority of subjects in both groups were male. Total IgG and IgG2 levels in lavage fluid were assayed in both cohorts and compared using a two-tailed Student's t test. RESULTS: The lung lining fluid IgG level in HIV-infected patients was 0.19 +/- 0.13 microg/microg of protein (mean +/- SD) vs 0.11 +/- 0.09 microg/microg of protein in control subjects (p < 0.05). The IgG(2) level in HIV patients was 0.034 +/- 0.038 microg/microg of protein and 0.014 +/- 0.01 microg/microg of protein in control subjects (p = 0.054). Lavage IgG levels reflected serum IgG values (correlation coefficient, 0.56; p < 0.001) but did not correlate with lung immunoglobulin-producing cells. CONCLUSIONS: The increased susceptibility to bacterial pneumonia in asymptomatic HIV-infected individuals is neither explained by depressed total IgG levels nor a deficiency in IgG(2) levels in the lungs. The strong correlation between serum and lavage IgG levels suggests that lavage IgG derives from serum.

ATP-stimulated release of interleukin (IL)-1beta and IL-18 requires priming by lipopolysaccharide and is independent of caspase-1 cleavage.

Interleukin (IL)-1beta and IL-18 are structurally similar proteins that require caspase-1 processing for activation. Both proteins are released from the cytosol by unknown pathway(s). To better characterize the release pathway(s) for IL-1beta and IL-18 we evaluated the role of lipopolysaccharide priming, of interleukin-1beta-converting enzyme (ICE) inhibition, of human purinergic receptor (P2X(7)) function, and of signaling pathways in human monocytes induced by ATP. Monocytes rapidly processed and released both IL-1beta and IL-18 after exogenous ATP. Despite its constitutive cytosolic presence, IL-18 required lipopolysaccharide priming for the ATP-induced release. Neither IL-1beta nor IL-18 release was prevented by ICE inhibition, and IL-18 release was not induced by ICE activation itself. Release of both cytokines was blocked completely by a P2X7 receptor antagonist, oxidized ATP, and partially by an antibody to P2X(7) receptor. In evaluating the signaling components involved in the ATP effect, we identified that the protein-tyrosine kinase inhibitor, AG126, produced a profound inhibition of both ICE activation as well as release of IL-1beta/IL-18. Taken together, these results suggest that, although synthesis of IL-1beta and IL-18 differ, ATP-mediated release of both cytokines requires a priming step but not proteolytically functional caspase-1.

Increased susceptibility to pulmonary emphysema among HIV-seropositive smokers.

BACKGROUND: Previous uncontrolled reports have suggested that HIV-seropositive persons develop an accelerated form of emphysema. OBJECTIVE: To characterize the risk for emphysema in a stable HIV-seropositive outpatient population. DESIGN: Controlled, cross-sectional analysis. SETTING: Midwestern urban community. PARTICIPANTS: HIV-seropositive persons (n = 114) without AIDS-related pulmonary complications and HIV-seronegative controls (n = 44), matched for age and smoking history. MEASUREMENTS: Measurement of pulmonary function, bronchoalveolar lavage, and high-resolution computed tomography of the chest. RESULTS: The incidence of emphysema was 15% (17 of 114) in the HIV-seropositive group compared with 2% (1 of 44) in the HIV-seronegative group (P = 0.025). The incidence of emphysema in participants with a smoking history of 12 pack-years or greater was 37% (14 of 38 persons) in the HIV-seropositive group compared with 0% (0 of 14 persons) in the HIV-seronegative group (P = 0.011). The percentage of cytotoxic lymphocytes in lavage fluid was much higher in HIV-seropositive smokers with emphysema. CONCLUSIONS: Infection with HIV accelerates the onset of smoking-induced emphysema. The results of this study support the emerging concept that cytotoxic lymphocytes may have an important role in emphysema pathogenesis.

Induction of interleukin-8 release by lung epithelium with cystic fibrosis epithelial lining fluid is marginally affected by inhibitors of interleukin-1beta.

Interleukin-1beta (IL-1beta) and neutrophil elastase (NE) are present in the epithelial lining fluid (ELF) of patients with cystic fibrosis (CF). Both factors activate surrounding cells including lung epithelial cells, causing release of IL-8, a potent chemoattractant for neutrophils. Previous studies showed up-regulation of IL-8 release by lung epithelial cells as a function of NE in CF; however, few studies addressed the relationship between IL-1beta and activation of lung epithelial cells in CF lungs. Confluent layers of A549 cells, a type II-like human lung epithelial cell line, were incubated overnight with IL-1beta (0-5 ng/ml) or NE (100 nM), and supernatants were analyzed for IL-8 by enzyme-linked immunosorbent assay (ELISA). Both IL-1beta and NE led to a significant increase in IL-8: 12.8 +/- 2.8 ng/ml and 0.8 +/- 0.3 ng/ml, respectively. Next, bronchoalveolar lavage (BAL) samples were obtained from one healthy adult volunteer and six patients with CF and measured for IL-8 and IL-1beta concentrations by ELISA. Both IL-8 (range 169.00 +/- 56.57 to 1742.04 +/- 338.98 pg/ml) and IL-1beta (range 0-24.26 +/- 0.52 pg/ml) were detected in CF specimens, whereas neither was detected in the volunteer's specimen. Normal and CF BALs then were incubated overnight at a 1:10 dilution with confluent A549 cells. Analysis by ELISA of cell-free supernatants revealed increased IL-8 production from cells stimulated with CF BALs only. Similar experiments were performed with BAL supernatants that had been incubated with soluble IL-1 type II receptor, soluble IL-1 receptor antagonist, or a peptide inhibitor of NE. Addition of IL-1 inhibitors had a marginal effect on the amount of IL-8 release after incubation with CF BAL samples, whereas inhibition of NE had no effect. Our results indicate that other factors present in ELF in CF account for IL-8 release from lung epithelial cells.

Spontaneous human monocyte apoptosis utilizes a caspase-3-dependent pathway that is blocked by endotoxin and is independent of caspase-1.

Apoptosis is an important mechanism for regulating the numbers of monocytes and macrophages. Caspases (cysteine-aspartate-specific proteases) are key molecules in apoptosis and require proteolytic removal of prodomains for activity. Caspase-1 and caspase-3 have both been connected to apoptosis in other model systems. The present study attempted to delineate what role these caspases play in spontaneous monocyte apoptosis. In serum-free conditions, monocytes showed a commitment to apoptosis as early as 4 h in culture, as evidenced by caspase-3-like activity. Apoptosis, as defined by oligonucleosomal DNA fragmentation, was prevented by a generalized caspase inhibitor, z-VAD-FMK, and the more specific caspase inhibitor, z-DEVD-FMK. The caspase activity was specifically attributable to caspase-3 by the identification of cleavage of procaspase-3 to active forms by immunoblots and by cleavage of the fluorogenic substrate DEVD-AFC. In contrast, a caspase-1 family inhibitor, YVAD-CMK, did not protect monocytes from apoptosis, and the fluorogenic substrate YVAD-AFC failed to show an increase in activity in apoptotic monocytes. When cultured with LPS (1 microgram/ml), monocyte apoptosis was prevented, as was the activation of caspase-3. Unexpectedly, LPS did not change baseline caspase-1 activity. These findings link spontaneous monocyte apoptosis to the proteolytic activation of caspase-3.

The pathophysiology of pulmonary diffusion impairment in human immunodeficiency virus infection.

Numerous reports have demonstrated that prior to the development of acquired immunodeficiency syndrome (AIDS)-related pulmonary complications, human immunodeficiency virus-positive (HIV+) individuals commonly develop unexplained reductions in pulmonary diffusing capacity (DLCO). The potential relevance of this observation is underscored by recent data demonstrating that reductions in DLCO independently predict the subsequent development of opportunistic pneumonia. To delineate the alterations in gas exchange associated with HIV, we investigated a group of HIV+ subjects with unexplained reductions in DLCO, using high-resolution computed tomography (HRCT) of the chest and a separation of diffusing capacity into its membrane (Dm) and capillary blood volume (Vc) components. We compared this abnormal group with HIV+ subjects with more normal gas exchange and also with a group of HIV- volunteers matched for age and smoking history. Compared with other groups, the HIV+ group with diffusion impairment demonstrated prominent reductions in Vc, despite a well-preserved total lung capacity (TLC). HRCT demonstrated virtually no evidence of interstitial fibrosis in any HIV+ subject, but evidence of early emphysema that significantly correlated with DLCO. Our results suggest that the previously reported impairment in pulmonary gas exchange in the HIV+ population involves loss of Vc and likely represents the development of early emphysema.

Regulation of monocyte survival in vitro by deposited IgG: role of macrophage colony-stimulating factor.

IgG deposition at tissue sites characteristically leads to macrophage accumulation and organ injury. Although the mechanism by which deposited IgG induces tissue injury is not known, we have recently demonstrated that deposited IgG stimulates the release of IL-8 and monocyte chemoattractant protein-1 from normal human monocytes, which may drive inflammation. Since IgG also induces macrophage accumulation in these diseases, we hypothesized that deposited IgG protects monocytes from apoptosis. As an in vitro model of the effect of deposited IgG on monocyte survival, monocyte apoptosis was studied after FcgammaR cross-linking. Monocytes cultured on immobilized IgG, which induces FcgammaR cross-linking, were protected from apoptosis, whereas monocytes cultured with equivalent concentrations of F(ab')2 IgG or 50 times higher concentrations of soluble IgG, neither of which induces FcgammaR cross-linking, were not protected. Moreover, this protection was transferable, as supernatants from immobilized IgG-stimulated monocytes protected freshly isolated monocytes from apoptosis and contained functional M-CSF, a known monocyte survival factor. M-CSF mediated the monocyte survival induced by FcgammaR cross-linking, as neutralizing anti-human M-CSF Abs blocked the monocyte protection provided by either immobilized IgG or IgG-stimulated monocyte supernatants. These findings demonstrate a novel mechanism by which deposited IgG targets tissue macrophage accumulation through FcgammaR-mediated M-CSF release. This pathway may play an important role in promoting and potentiating IgG-mediated tissue injury.

Extracellular regulation of interleukin (IL)-1beta through lung epithelial cells and defective IL-1 type II receptor expression.

Interleukin (IL)-1beta is produced primarily by activated mononuclear phagocytic cells in the lung airway and functions as a potent proinflammatory cytokine. Release of IL-1beta in the airway microenvironment induces the production of proinflammatory factors from parenchymal airway cells, including IL-8. To study the regulation of lung epithelial cell responsiveness to IL-1beta, the human type II-like airway epithelial cell line A549 and primary normal human bronchial epithelial (NHBE) cells were assayed for IL-1-specific response modifiers. Specifically, the IL-1 type I receptor (IL-1RI), IL-1 type II receptor (IL-1RII), IL-1 receptor accessory protein (IL-1RAcP), and IL-1 receptor antagonist (IL-1Ra) were analyzed. Constitutive expression of IL-1RI, IL-1RAcP, and IL-1Ra was detected in both immortalized and primary human airway epithelial cells. Interestingly, a complete absence of IL-1RII expression was demonstrated under all study conditions in both A549 and NHBE cells. Both cell types were responsive to IL-1beta at concentrations as low as 50 to 500 pg/ml when measured by IL-8 release into cell supernatants. IL-1beta-induced chemokine production and release were inhibited by a 10- to 1,000-fold molar excess of recombinant IL-1RII or IL-1Ra, whereas IL-1RI was a less effective inhibitor. On the basis of our results, we propose that human lung epithelial cells lack the ability to downregulate IL-1beta activity extracellularly because of an inability to express IL-1RII. Release of extracellular IL-1 inhibitors, including soluble IL-1Ra and soluble IL-1RII, by other inflammatory cells present in the airway may be critical for regulation of IL-1beta activity in the airway microenvironment.

Endotoxin-stimulated monocytes release multiple forms of IL-1 beta, including a proIL-1 beta form whose detection is affected by export.

The processing and release of 31-kDa proIL-1 beta to the mature 17-kDa form of IL-1 beta are still poorly understood. To help elucidate the mechanisms involved in IL-1 beta processing and release, we measured IL-1 beta forms released from endotoxin-stimulated monocytes by immunoprecipitation of [35S]methionine-labeled protein, by Western blots, and by our recently developed ELISA specific for proIL-1 beta. Our studies demonstrate that in addition to the 17-kDa mature IL-1 beta, IL-1 beta is also released as 31-, 28-, and 3-kDa molecules. The 31-kDa-released form of proIL-1 beta represented 20-40% of the total released IL-1 beta, as measured by SDS-PAGE with densitometry. This released proIL-1 beta was susceptible to ICE processing; however, this proIL-1 beta was not detectable by either a mature or proIL-1 beta-specific ELISA, suggesting that release induces a conformational change. The ELISA inability to detect proIL-1 beta was not due to inadequate sensitivity or subsequent degradation in the ELISA. Furthermore, while immunoaffinity-purified cytosolic proIL-1 beta could complex the type II IL-1R, released proIL-1 beta did not. Finally, the absence of a band shift in nondenaturing gel electrophoresis excluded proIL-1 beta binding to another protein. These findings imply that IL-1 beta is exported from monocytes as 3-, 17-, 28-, and 31-kDa forms and that the released 31-kDa form differs from cytosolic proIL-1 beta.

Cigarette smoking in HIV infection induces a suppressive inflammatory environment in the lung.

Lung lymphocyte numbers are frequently increased in human immunodeficiency virus (HIV)-infected individuals in the absence of lung infection, and may play a critical role in viral surveillance and protection against new infections. In this context, cigarette smoking by HIV-infected individuals has been associated with a relative increase in the peripheral blood CD4(+) T-lymphocyte count as compared with that of nonsmokers. Because lung defense is local, the aim of the present study was to determine whether cigarette smoking had a significant impact on local lung defenses in HIV-infected individuals. The numbers and subtypes of bronchoalveolar lymphocytes and the ability of lung lavage cells to produce proinflammatory cytokines were compared in 58 smokers and 34 nonsmokers. In contrast to a trend toward an increase in peripheral blood CD4(+) cell counts among nonsmokers, smokers had significant depressions in both the percentage and absolute numbers of CD4(+) and CD8(+) cells in their bronchoalveolar lavage fluid (BALF). A decrease in CD4(+)/CD8(+) cell ratios was also seen with smoking. In addition, production of both interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) was suppressed with cigarette smoking. These observations show that cigarette smoking is associated with suppression in localized lung defenses, and suggest that smoking cessation may have a positive impact on lung defenses in HIV-infected smokers.

Lymphocytes produce IL-1beta in response to Fcgamma receptor cross-linking: effects on parenchymal cell IL-8 release.

Neutrophils mediate tissue injury in response to immune complexes, although the factors that induce their recruitment are incompletely understood. We have reported that lymphocytes may be important regulators of monocyte and macrophage IL-8 release in the presence of immobilized IgG. Since tissue parenchymal cells are important local producers of IL-8 but are not directly stimulated by FcgammaR cross-linking, we hypothesized that lymphocytes may also regulate parenchymal IL-8 release. Supernatants from lymphocytes incubated on immobilized IgG induced primary human fibroblasts and human mesangial cells to produce IL-8 (17 +/- 3.5 and 44 +/- 8 ng/ml, respectively). Fibroblast and mesangial cell IL-8 mRNA levels were similarly increased by the conditioned lymphocyte supernatant. Immobilized anti-human FcgammaRIII, but not FcgammaRI or FcgammaRII Abs, could stimulate this IL-8-inducing activity in lymphocytes, suggesting that FcgammaRIII-bearing lymphocytes were responsible. Supernatants from lymphocytes incubated on immobilized IgG contained 2.2 +/- 0.8 ng/ml of IL-1beta, while enriched monocyte preparations from the same donors incubated on immobilized IgG released only 0.1 +/- 0.04 ng/ml of IL-1beta (p = 0.05). Consistent with the identification of IL-1beta as the lymphocyte factor, fibroblast or mesangial cell IL-8 release induced by the IgG-stimulated lymphocyte supernatants was inhibited by 1) the combination of IL-1R antagonist and soluble type II IL-1R, 2) an IL-1-converting enzyme inhibitor, or 3) anti-IL-1beta but not preimmune Abs. These data suggest that targeted deposits of IgG can stimulate FcgammaRIII-bearing lymphocytes to produce IL-1beta, which induces parenchymal cell IL-8 release.

Alpha 1-antitrypsin and protease complexation is induced by lipopolysaccharide, interleukin-1beta, and tumor necrosis factor-alpha in monocytes.

Local regulation of alpha1-antitrypsin (alpha1-AT) may have importance in maintenance of the protease-antiprotease balance in the microenvironment of inflammatory cells. We therefore studied whether lipopolysaccharide (LPS), interleukin-1beta (IL-1beta), and tumor necrosis factor-alpha (TNFalpha) affect the pericellular concentration of alpha1-AT in human peripheral blood mononuclear cells (PBMC). PBMC taken from normal healthy volunteers were treated with LPS, IL-1beta, and TNFalpha, and the concentration of human alpha1-AT in conditioned supernatants was measured. When compared with unstimulated control supernatants (147 +/- 19 ng/ml), LPS (439 +/- 66 ng/ml; p < or = 0.001), IL-1beta (263 +/- 37 ng/ml; p < or = 0.01), and TNFalpha (316 +/- 59 ng/ml; p < or = 0.05) induced a 2- to 3-fold increase of alpha1-AT. Up-regulation of alpha1-AT protein correlated with an increase in alpha1-AT mRNA, suggesting a simultaneous increase in alpha1-AT synthesis. Despite the increase in alpha1-AT concentration, functional antiprotease activity could not be detected. Furthermore, protease activity was present in all samples, with the amount of activity being inversely related to the amount of alpha1-AT measured in supernatants. These findings suggest that local inflammatory conditions up-regulate alpha1-AT production by monocytes which complex with a protease derived from the PBMC population.

Antineutrophil cytoplasmic antibodies induce monocyte IL-8 release. Role of surface proteinase-3, alpha1-antitrypsin, and Fcgamma receptors.

Cytoplasmic antineutrophil cytoplasmic antibodies (cANCA) that accompany the neutrophilic vasculitis seen in Wegener's granulomatosis (WG), are directed against proteinase-3 (PR-3), a serine proteinase which is located in azurophilic granules of neutrophils and monocytes. PR-3, when expressed on the surface of TNFalpha-primed neutrophils, can directly activate neutrophils by complexing cANCA and promoting concomitant Fcgamma receptor (FcgammaR) cross-linking. Although the neutrophil's pathogenic role in WG has been studied, the role of the monocyte has not been explored. The monocyte, with its ability to release cytokines and regulate neutrophil influx, also expresses PR-3. Therefore, the monocyte may play a significant role in WG via the interaction of surface PR-3 with cANCA, inducing cytokine release by the monocyte. To test this hypothesis, monocytes were studied for PR-3 expression and for IL-8 release in response to cANCA IgG. PBMC obtained from healthy donors displayed dramatic surface PR-3 expression as detected by immunohistochemistry and flow cytometry in response to 0. 5-h pulse with TNFalpha (2 ng/ml). Purified monoclonal anti-PR-3 IgG added to TNFalpha-primed PBMC induced 45-fold more IL-8 release than an isotype control antibody. Furthermore, alpha 1-antitrypsin (alpha1-AT), the primary PR-3 antiprotease, inhibited the anti-PR-3 induced IL-8 release by 80%. Importantly, Fab and F(ab')2 fragments of anti-PR-3 IgG, which do not result in Fcgamma receptor cross-linking, do not induce IL-8 release. As a correlate, IgG isolated from cANCA positive patients with WG induced six times as much PBMC IL-8 release as compared to IgG isolated from normal healthy volunteers. Consistent with PR-3 associated IL-8 induction, alpha1-AT significantly inhibited this effect. These observations suggest that cANCA may recruit and target neutrophils through promoting monocyte IL-8 release. This induction is mediated via Fcgamma receptor cross-linking and is regulated in part by alpha1-AT.

Changes in mononuclear phagocyte microtubules after endotoxin stimulation. I. Changes in microtubule stability.

Microtubules are in a dynamic equilibrium of polymerization and depolymerization. In monocytes and macrophages, microtubules bind endotoxin and partly regulate inflammatory events such as cytokine production. To characterize the morphologic differences between alveolar macrophage and blood monocyte microtubules after LPS stimulation, cells were examined by immunofluorescent microscopy and laser confocal microscopy. Fresh monocytes contained an average of 26 microtubules per cell which significantly increased to 31 microtubules per cell following a 30-min exposure to LPS (P < 0.001). Using a nocodazole-based assay of microtubule dynamic instability, the half-life of fresh unstimulated human monocyte microtubules was approximately 18 s and extended to 26 s following a 30-min exposure to LPS. In vitro maturation of monocytes for 18 h increased microtubule stability but not number. Compared to monocytes, alveolar macrophage microtubules were longer, more numerous, and much more stable. These results suggest that alveolar macrophage microtubules are more numerous and stable than blood monocyte microtubules and that LPS causes an increase in monocyte microtubule number and stability.

Changes in mononuclear phagocyte microtubules after endotoxin stimulation. II. Changes in microtubule composition.

Microtubules are integral components of the cytoskeleton of human cells and are composed of alpha- and beta-tubulin as well as a variable number of microtubule-associated proteins. In monocytes and macrophages, microtubules bind endotoxin and partly regulate endotoxin-induced inflammatory events such as cytokine production. Endotoxin causes a rapid alteration in monocyte microtubule stability. To characterize the effect of endotoxin on mononuclear phagocyte microtubule composition, Western blots and flow cytometry were performed on human monocytes and the monocyte/macrophage-like cell line THP-1. Compared to unstimulated monocytes, monocytes stimulated with endotoxin for 18 h had increased quantities of alpha-, beta-, and tyrosinated alpha-tubulin as well as microtubule-associated protein-2. PMA-differentiated THP-1 cells had increased levels of alpha-tubulin, beta-tubulin, microtubule-associated protein-5, microtubule-associated protein-2, and tau after endotoxin stimulation. These results indicate that endotoxin can alter mononuclear phagocyte microtubules by causing an increase in certain microtubule component proteins.